1. Field of the Invention
The present invention relates generally to a unique mammalian cell line expressing inducible c-Src, and, particularly, a unique human cell line overexpressing c-Src in an inducible manner.
2. Description of the Related Art
Macropinocytosis enables nonselective uptake of solute macromolecules, including nutrients and antigens, from the extracellular milieu through formation of clathrin-independent large (>1 μm in diameter) vesicles of irregular size (Swanson and Watts, 1995; Cardelli 2001; Conner and Schmid, 2003). Although high macropinocytic activity is observed in phagocytes such as macrophages and dendritic cells, most cells do not exhibit macropinocytic activity under normal culture conditions (Swanson and Watts, 1995). Some cell types show remarkable macropinocytosis after stimulation with growth factors, such as macrophage colony-stimulating factor (M-CSF) and epidermal growth factor (EGF) (Racoosin and Swanson, 1993; Hewlett et al., 1994), or mitogens such as phorbol 13-myristate 12-acetate (PMA) (Swanson, 1989; Sun et al., 2003; Sun and Endo, 2005).
Macropinosomes are generated from sites of membrane ruffling, which can take two forms: cell edge ruffling and dorsal surface ruffling (Swanson and Watts, 1995; Araki et al., 1996; Ellerbroek et al., 2004). After formation and subsequent budding, macropinosomes mature differently in different cell types. In macrophages, they mature with centripetal movement and shrinkage accompanied by a change in composition; a newly formed macropinosome begins as an early endosome derived from the plasma membrane, rapidly matures into a late endosome, and then merges with the lysosomal compartment, which is the end point of macropinocytosis (Racoosin and Swanson, 1992, 1993; Swanson and Watts, 1995). Unlike macrophages, in EGF-stimulated human epidermoid carcinoma A431 cells. macropinosomes are recycled back to the cell surface without fusing with lysosomes (Hewlett et al., 1994). However. little is known about how different macropinosomal maturation processes are regulated.
Src-family tyrosine kinases (SFKs), which are nonreceptor-type tyrosine kinases, consist of proto-oncogene products and structurally related proteins and include at least eight highly homologous proteins: c-Sre, Lyn, Fyn, c-Yes, c-Fgr, Hck, Lck, and Blk. SFKs regulate cell proliferation, migration, and cytoskeletal reorganization (Brown and Cooper, 1996; Thomas and Brugge, 1997). They also control membrane trafficking, such as endocytic internalization of the EGF receptor (EGFR), early endosomal dynamics, and organization of the Golgi apparatus to transport proteins to the ER (Ware at al., 1997; Bard et al., 2003; Gasman et al., 2003).
SFKs exhibit four highly conserved Src homology (SH) domains: an N-terminal SH4 domain (followed by a poorly conserved “Unique” domain), an SH3 domain, an SH2 domain, and an SH1 tyrosine kinase domain (Brown and Cooper, 1996, Thomas and Brugge, 1997). The SH4 domain contains the signal(s) for acylation, such as N-myristoylation and palmitoylation, and regulates association with the cytoplasmic surface of membranes, while SH3 and SH2 domains mediate protein-protein interactions.
Given the high homology of SFKs. it is not surprising that their functions overlap. Although mice or cells deficient in multiple members of the SFK family display more severe defects than those lacking a single SFK (Stein et al., 1994; Thomas and Brugge, 1997; Bard et al., 2003), specific functions of single SFKs have been noted in knockout mice or cells (Thomas et at., 1995; Lowell et al., 1996; Thomas and Brugge, 1997). However, it is unclear how the functional specificity of a specific SFK is determined.